The 26S proteasome is a massive, intricately regulated ATP-dependent protease that is responsible for the degradation of most cellular proteins. Failure of the proteasome to precisely regulate protein levels is a hallmark of many cancers and neurodegenerative diseases. Targeting proteins to the proteasome requires covalent modification with a polymeric (Ub) chain. Thus, it is perplexing that the proteasome actually houses enzymes (deubiquitinases/DUBs) responsible for removing Ub chains. While it has become clear that the intrinsic DUB, RPN11, promotes degrades by preventing premature deubiquitination of proteins not yet engaged with the proteasome, the roles of other proteasomal DUBs, e.g., UCH37/UCHL5, are poorly understood. In preliminary studies, we discovered that proteasome-bound UCH37 acts as a chain editor by removing branch points. This application proposes to elucidate how UCH37 selects branched Ub chains for editing, how this activity is integrated into the entire process of proteasomal degradation, and how it impacts the turnover of cellular proteins.
In Aim 1, we will investigate the role of UCH37-mediated chain debranching during proteasomal degradation using distinct, purified human proteasome complexes and fluorescent, polyubiquitinated substrates.
In Aim 2, we propose to identify cellular targets of proteasome-bound UCH37 using an innovative combination of quantitative proteomics, in-cell proximity labeling, and Ub middle-down mass spectrometry.
In Aim 3, we focus on understanding the molecular basis of UCH37's specificity toward branched chains. Finally, in Aim 4, we will develop novel cyclic peptide inhibitors of UCH37 to facilitate efforts to dissect its function in any biological paradigm. The knowledge gained from this research will shed light on fundamental aspects of the ubiquitin proteasome system and pave the way for the development of new therapeutics that regulate proteasome function.
This proposal aims to understand how a proteasome-associated deubiquitinase works to regulate protein degradation. Our recent discovery that proteasome-bound ubiquitin chains are edited by removing branch points suggests chain debranching plays an important role in the degradation process, and in this applica- tion we will test this hypothesis. The translation of our fundamental insights into small molecule modulators is especially important as many disease-related pathways are regulated by aberrant proteasome function.
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